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- Materials and methods
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Equine metabolic syndrome is associated with hyperinsulinaemia and insulin resistance, and in these horses insulin resistance may be a predisposing factor for laminitis . Significantly lower plasma insulin concentrations were found in healthy ponies than in ponies that later developed laminitis . Experimentally, clinical laminitis was successfully induced in ponies and Standardbreds after 48 h of insulin infusion leading to serum concentrations >1000 μiu/ml [3, 4]. This confirmed insulin as one of several primary causes for the development of equine laminitis. In a study more closely reflecting the natural course of disease progression, 48 h of hyperglycaemia with resultant hyperinsulinaemia (serum concentrations >200 μiu/ml) also led to laminitic changes in lamellar histology .
While several aspects of the relationship between insulin and laminitis have been investigated, the underlying pathological pathway has not yet been identified. Insulin has both vasodilatory and vasoconstrictive effects on endothelial function . In the endothelium of healthy human subjects, insulin binds to insulin receptors, which leads to the phosphorylation of insulin receptor substrate-1, resulting in activation of endothelial nitric oxide synthase via the phosphoinositide 3-kinase pathway. The subsequent increase of nitric oxide secretion results in vasodilatation. This pathway is also involved in the metabolic regulatory action of insulin. In contrast, a study in insulin-resistant mice showed that insulin can lead to activation of a mitogen-activated protein (MAP)-kinase pathway, with an increase in the secretion and activation of the vasoconstrictor endothelin-1 (ET-1) [7, 8].
In insulin-resistant rats, impairment of the phosphoinositide 3-kinase pathway leads to an imbalance, with vasoconstriction outweighing vasodilatation . This vasoconstriction was recently also shown in insulin-resistant explants of equine digital veins and arteries. In both types of vessels, insulin resistance led to a further constriction in response to insulin. This effect was blocked by a specific MAP-kinase blocker, identifying its MAP-kinase dependency . When exposed to the same concentrations of ET-1, laminar veins constricted more than laminar arteries . The resulting pressure differences between the pre- and post capillary beds were hypothesised to contribute to changes in the digital Starling forces observed in horses with clinical laminitis .
In order to investigate the development of laminitis in a semi-complex system, ex vivo perfusion of equine digits has recently been established [11, 12]. This model fills the considerable gap between tissue cultures, vessel explants and in vivo tests, with a research focus on glucose and insulin metabolism, oxygen consumption and perfusion pressure. Earlier, a model of Krebs–Henseleit-perfused equine digits had been used to assess the responsiveness of the vascular system of the equine digit .
The aim of the present study was to monitor the effect of short-term (9 h) hyperinsulinaemia on the perfused isolated equine digit. The following hypotheses were investigated.
- During hyperinsulinaemic perfusion, digital vascular resistance is higher than during normoinsulinaemic perfusion.
- After hyperinsulinaemic perfusion, expression of ET-1 in the laminar tissue is higher than after normoinsulinaemic perfusion.
- Top of page
- Materials and methods
- Authors’ declaration of interests
- Source of funding
We used the model of extracorporeal perfusion in the isolated equine digit to investigate metabolic variables. This model has several drawbacks, and results cannot be transferred to the in vivo situation without considering the major differences between the experimental set-up and the complex in vivo situation. The lack of hepatic, pancreatic and neural functions in the model must be assumed to influence the metabolism of glucose and insulin and its effect on the vascular system. In several recent studies, other in vitro and ex vivo models (vessel explants, Krebs–Henseleit perfusion models) have been successfully employed to increase the knowledge about the effect of ET-1 and insulin on the equine digital vascular system [6, 9, 16].
In the present study, hyperinsulinaemia did not change the glucose metabolism within the 9 h of perfusion. This finding supports a recent finding that glucose metabolism of the lamellar tissue is mainly insulin independent . In the equine digit, a number of tissue types are present, with the lamellar tissue having the highest glucose use compared with the other tissues, such as tendon and skin . The overall glucose consumption of the digit can therefore be assumed to be directly associated with the glucose metabolism of the lamellar tissue.
Other substrates were chosen to monitor anaerobic glycolysis (lactate) and possible cell destruction during the perfusion (LDH). Generation of LDH and lactate had a large variation and was not significantly different between the CP group and the IP group, while concentration values showed less variation, with the concentration of LDH being significantly lower and the concentration of lactate significantly higher in the IP group. This finding is in agreement with rodent studies, in which application of insulin led to a decrease in LDH concentration and an increase in lactate concentration, documented to be the consequence of a reduced rate of apoptosis .
In the present study, ET-1 was investigated as one of several possible factors influencing vascular resistance. Immunohistochemistry was chosen to detect ET-1 in the laminar tissue, because it was shown that endothelial cells secrete 80% of ET-1 abluminally in the direction of the tunica media . This method has successfully been used in kidney tissue of human patients with type 2 diabetes, where its higher expression compared with healthy control subjects was proved . One study on ET-1 expression has used plasma concentration to monitor ET-1 in clinically endotoxaemic horses ; however, this was not feasible in the present study owing to the need for repeated reservoir exchanges.
The increased ET-1 expression seen in the present study could reflect an activation of the MAP-kinase pathway following hyperinsulinaemia. This activation effect was previously shown in vessel explants, which developed insulin resistance after 30 min of incubation with 10 mmol/l insulin . In the present study, insulin concentrations were much lower, at about 0.8 mmol/l, and ET-1 expression was increased after 9 h of insulinaemia; however, the MAP-kinase pathway was not documented. Insulin concentrations in the present study were within the range of those reported in laminitis-prone horses with naturally occurring hyperinsulinaemia (69.5 ± 19.8 μiu/ml) and in ponies with clinical laminitis (>100 μiu/ml) [23, 24]. Although a set amount of insulin was added to each perfusate in the IP group, there were large interindividual variations in mean insulin concentration in the present study. This may be explained by the use of limbs from a large variety of donor horses, given that factors such as sex, breed, age and exercise level are known to influence the insulin-binding ability of blood cells and tissues.
Endothelin-1 might play only a small part in the influence of insulin on the vascular resistance. This study did not investigate a possible reduction in the expression of nitric oxide . Insulin-induced activation of the MAP-kinase pathway might also lead to an increased expression of vascular cell adhesion molecules and E-selectin, increasing vascular resistance . Changes in the sensitivity to other vasoconstrictive agents, such as 5-hydroxytryptamine or thromboxane, are also discussed in the development of laminitis, but these were not assessed in the present study [26, 27]. Besides these changes in the reactivity of the vessels to different agents, the specific microarchitectural details, such as arteriovenous shunts, might react to hyperinsulinaemia and influence the vascular resistance.
In general, vascular resistance is affected by the balance between vasodilatory and vasoconstrictive effects, and in this model this was calculated from the perfusion pressure during a steady perfusion flow . The increase of vascular resistance in the IP group might reflect a vasoconstriction of the digital vessels and, from earlier studies in vascular explants, this is thought to be due to the high sensitivity of veins to ET-1 . The arteriovenous pressure difference in the presence of ET-1 and the proposed high post capillary resistance may have caused the formation of oedema surrounding the vessels in the IP group limbs of the present study . No such oedema was detected in the CP group or in previously investigated endotoxaemic conditions [11, 12]. In experimental in vivo laminitis after hyperinsulinaemia, no oedema formation was noted after 48 h; this is in contrast to the present study . A possible explanation for this difference might be the pressure conditions within the hoof capsule, because the digits in the present study remained unloaded, while the horses in the in vivo experiment loaded and unloaded the hooves at stance and during weight shifting in early laminitis, creating an increased backflow. Other reasons for the oedema formation seen in the present study might be a mild vasculitis, which might have become apparent using electron microscopy even though this was not noted on light microscopy. Decreased oncotic pressure within the vessels could also have occurred even though the total protein concentrations remained stable over the 10 h of perfusion. Using histology, it cannot be determined whether formation of oedema within the hoof capsule led to an increased vascular resistance or whether the vascular resistance resulting from changed vessel tone led to the formation of oedema.
The increased vascular resistance seen in the present study could be represented by increased blood pressure in the live horse. This was shown in prelaminitic ponies, which had higher blood pressure values (measured in the coccygeal artery) in comparison with healthy ponies . However, a direct comparison between the model and the in vivo blood pressure values is not possible because influences on the blood pressure in the live horse (e.g. pain) cannot be reproduced in the model. Additionally, increased resistance in the lamellar tissue alone may not be sufficient to raise blood pressure overall. In future, the recently published method of measuring blood pressure in equine digit may be useful to permit better comparison of in vivo and perfusion model results in hyperinsulinaemic and healthy horses .
Short-term hyperinsulinaemia in the isolated, perfused extracorporeal equine digit leads to a marked increase in vascular resistance and an increase in ET-1 expression. Increased vascular resistance might be a result of increased ET-1 expression. However, more data are needed in order to understand the relationship between blood pressure, ET-1 and laminitis fully, while additional in vivo experiments are necessary to prove the suitability of the model.